Multi-Tissue Transcriptome Analysis Identifies Key Sexual Development-Related Genes of the Ornate Spiny Lobster (Panulirus ornatus).

Sexual development involves the successive and overlapping processes of sex determination, sexual differentiation, and ultimately sexual maturation, enabling animals to reproduce. This provides a mechanism for enriched genetic variation which enables populations to withstand ever-changing environments, selecting for adapted individuals and driving speciation. The molecular mechanisms of sexual development display a bewildering diversity, even in closely related taxa. Many sex determination mechanisms across animals include the key family of "doublesex- and male abnormal3-related transcription factors" (Dmrts). In a few exceptional species, a single Dmrt residing on a sex chromosome acts as the master sex regulator. In this study, we provide compelling evidence for this model of sex determination in the ornate spiny lobster Panulius ornatus, concurrent with recent reports in the eastern spiny lobster Sagmariasus verreauxi. Using a multi-tissue transcriptomic database established for P. ornatus, we screened for the key factors associated with sexual development (by homology search and using previous knowledge of these factors from related species), providing an in-depth understanding of sexual development in decapods. Further research has the potential to close significant gaps in our understanding of reproductive development in this ecologically and commercially significant order.

Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1, NOP10, or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin-NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin-NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.

Mutations in LAMB2 Are Associated With Albuminuria and Optic Nerve Hypoplasia With Hypopituitarism.

CONTEXT: Mutations in LAMB2, encoding the basement membrane protein, laminin ß2, are associated with an autosomal recessive disorder characterized by congenital nephrotic syndrome, ocular abnormalities, and neurodevelopmental delay (Pierson syndrome). CASE DESCRIPTION: This report describes a 12-year-old boy with short stature, visual impairment, and developmental delay who presented with macroscopic hematuria and albuminuria. He had isolated growth hormone deficiency, optic nerve hypoplasia, and a small anterior pituitary with corpus callosum dysgenesis on his cranial magnetic resonance imaging, thereby supporting a diagnosis of optic nerve hypoplasia syndrome. Renal histopathology revealed focal segmental glomerulosclerosis. Using next-generation sequencing on a targeted gene panel for steroid-resistant nephrotic syndrome, compound heterozygous missense mutations were identified in LAMB2 (c.737G>A p.Arg246Gln, c.3982G>C p.Gly1328Arg). Immunohistochemical analysis revealed reduced glomerular laminin ß2 expression compared to control kidney and a thin basement membrane on electron microscopy. Laminin ß2 is expressed during pituitary development and Lamb2-/- mice exhibit stunted growth, abnormal neural retinae, and here we show, abnormal parenchyma of the anterior pituitary gland. CONCLUSION: We propose that patients with genetically undefined optic nerve hypoplasia syndrome should be screened for albuminuria and, if present, screened for mutations in LAMB2.

Y-linked iDmrt1 paralogue (iDMY) in the Eastern spiny lobster, Sagmariasus verreauxi: The first invertebrate sex-linked Dmrt.

Sex determination pathways are extensively diverse across species, with the master sex-determinants being the most variable element. Despite this, there is a family of DM-domain transcription factors (Dmrts), which hold a highly conserved function in sexual development. This work is the first to describe a heterogametic sex-linked Dmrt in an invertebrate species, the Eastern spiny lobster, Sagmariasus verreauxi. We have termed the Y-linked, truncated paralogue of the autosomal iDmrt1, Sv-iDMY. Considering the master sex-determining function of both DMY in medaka and DM-W in frog, we hypothesised a similar function of Sv-iDMY. By conducting temporal expression analyses during embryogenesis we have identified a putative male sex-determining period during which iDMY>iDmrt1. Employing a GAL4-transactivation assay we then demonstrate the dominant negative suppression of iDMY over its autosomal iDmrt1 paralogue, suggesting the mechanism with which iDMY determines sex. Comparative analyses of Sv-iDMY, DM-W and medaka DMY, highlight the C'-mediated features of oligomerisation and transactivation as central to the mechanism that each exerts. Indeed, these features may underpin the plasticity facilitating the convergent emergence of these three sporadic sex-linked master-Dmrts.

Understanding Insulin Endocrinology in Decapod Crustacea: Molecular Modelling Characterization of an Insulin-Binding Protein and Insulin-Like Peptides in the Eastern Spiny Lobster, Sagmariasus verreauxi.

The insulin signalling system is one of the most conserved endocrine systems of Animalia from mollusc to man. In decapod Crustacea, such as the Eastern spiny lobster, Sagmariasus verreauxi (Sv) and the red-claw crayfish, Cherax quadricarinatus (Cq), insulin endocrinology governs male sexual differentiation through the action of a male-specific, insulin-like androgenic gland peptide (IAG). To understand the bioactivity of IAG it is necessary to consider its bio-regulators such as the insulin-like growth factor binding protein (IGFBP). This work has employed various molecular modelling approaches to represent S. verreauxi IGFBP and IAG, along with additional Sv-ILP ligands, in order to characterise their binding interactions. Firstly, we present Sv- and Cq-ILP2: neuroendocrine factors that share closest homology with Drosophila ILP8 (Dilp8). We then describe the binding interaction of the N-terminal domain of Sv-IGFBP and each ILP through a synergy of computational analyses. In-depth interaction mapping and computational alanine scanning of IGFBP_N' highlight the conserved involvement of the hotspot residues Q67, G70, D71, S72, G91, G92, T93 and D94. The significance of the negatively charged residues D71 and D94 was then further exemplified by structural electrostatics. The functional importance of the negative surface charge of IGFBP is exemplified in the complementary electropositive charge on the reciprocal binding interface of all three ILP ligands. When examined, this electrostatic complementarity is the inverse of vertebrate homologues; such physicochemical divergences elucidate towards ligand-binding specificity between Phyla.

Applying the Power of Transcriptomics: Understanding Male Sexual Development in Decapod Crustacea.

The decapod Crustacea are the most species-rich order of the Crustacea and include some of the most charismatic and highly valued commercial species. Thus the decapods draw a significant research interest in relation to aquaculture, as well as gaining a broader understanding of these species' biology. However, the diverse physiology of the group considered with the lack of a model species have presented an obstacle for comparative analyses. In reflection of this, the recent integration of comparative transcriptomics has rapidly advanced our understanding of key regulatory pathways and developmental phenomena, an example being our understanding of sexual development. We discuss our work in the Eastern spiny lobster, Sagmariasus verreauxi, in the context of what is currently known about male sexual development in the decapods, highlighting the importance of transcriptomic techniques in achieving our recent advancements. We describe the progression made in our understanding of male sexual differentiation and maturation, as mediated by the insulin-like androgenic gland hormone (IAG), integrating the role of regulatory binding proteins (IGFBPs), a tyrosine kinase insulin receptor (TKIR), as well as the upstream effect of neuroendocrine hormones (GIH and MIH). We then consider the less well understood mechanism of male sex determination, with an emphasis on what we believe to be the key regulatory factors, the Dsx- and mab-3-related transcription factors (Dmrts). Finally, we discuss the function of the antennal gland (AnG) in sexual development, relating to the emergence of male-biased upregulation in the AnG in later sexual maturation and the sexually dimorphic expression of two key genes Sv-TKIR and Sv-Dmrt1 We then present the AnG as a case study to illustrate how comparative transcriptomic techniques can be applied to guide preliminary analyses, like the hypothesis that the AnG may function in pheromone biosynthesis. In summary, we describe the power of transcriptomics in facilitating the progress made in our understanding of male sexual development, as illustrated by the commercial decapod species, S. verreauxi Considering future directions, we suggest that the integration of multiple omics-based techniques offers the most powerful tool to ensure we continue to piece together the biology of the important group of decapod Crustacea.

Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans.

Male Sexual Development and the Androgenic Gland: Novel Insights through the de novo Assembled Transcriptome of the Eastern Spiny Lobster, Sagmariasus verreauxi.

The Eastern spiny lobster, Sagmariasus verreauxi, is commercially important in fisheries, with growing aquaculture potential, driving an interest to better understand male sexual differentiation. Amongst the Decapoda, the androgenic gland (AG) and the insulin-like androgenic gland hormone (IAG) have a well-defined function in male sexual differentiation. However, IAG is not a sex determinant and therefore must be considered as part of a broader, integrated pathway. This work uses a transcriptomic, multi-tissue approach to provide an integrated description of male-biased expression as mediated through the AG. Transcriptomic analyses demonstrate that IAG expression is stage- and eyestalk-regulated (low in immature, high in mature and 6-times higher in hypertrophied glands), with IAG being the predominant AG-specific factor. The low expression of this key factor in immature males suggests the involvement of other tissues in male sexual differentiation. Across tissues, the gonad (87.8%) and antennal gland (73.5%) show the highest male-biased differential expression of transcripts and also express 4 sex-determination regulators, known as Dmrts, with broader expression of Sv-Sxl and Sv-TRA-2. In order to better understand male sexual differentiation, tissues other than the AG must also be considered. This research highlights the gonad and antennal gland as showing significant male-biased expression patterns, including the Sv-Dmrts.

Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi.

This study reports, for the first time in any of the commercially important decapod species, the identification of an insulin-like peptide (ILP), distinct from the androgenic gland hormone. Bioinformatics analysis of the de novo assembled spiny lobster, (Sagmariasus verreauxi) transcriptome, allowed identification of Sv-ILP1 as well as eight binding proteins. Binding proteins were termed as Sv-IGFBP, due to homology with the vertebrate insulin-like growth-factor binding protein and Sv-SIBD1-7, single insulin-binding domain protein (SIBD), similar to those identified in other invertebrate species. Sv-ILP1 was found to be expressed in the eyestalk, gonads and antennal gland of both sexes and to a lesser extent in male muscle, androgenic gland and hepatopancreas. The expression profiles of each binding protein were found to vary across tissues, with Sv-SIBD5, 6 and 7 showing higher expression in the gonad, demonstrated by PCR and digital gene expression. Further spatial investigations, using in-situ hybridisation, found Sv-ILP1 to be expressed in the neurosecretory cells of the thoracic ganglia, in keeping with the tissue expression of Drosophila ILP7 (DILP7). This correlative tissue expression, considered with the phylogenetic clustering of Sv-ILP1 and DILP7, suggests Sv-ILP1 to be a DILP7 orthologue. The broad expression of Sv-ILP1 strongly suggests that ILPs have a role beyond that of masculinisation in decapods. The function of these novel peptides may have application in enhancing aquaculture practices in the commercially important decapod species.